The synthetic triterpenoid (CDDO-Im) inhibits STAT3, as well as IL-17, and improves DSS-induced colitis in mice.

INTRODUCTION: Synthetic triterpenoids inhibit IL-17 and improve autoimmune disease in mice. A prototype triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im), also inhibits signal transducer and activator of transcription 3 (STAT3) activation. AIMS: The goals of our study were twofold: (1) To determine if ex vivo treatment with CDDO-Im attenuated colonic IL-17 secretion from isolated splenocytes and colonic strips; (2) To determine if oral treatment with CDDO-Im improved DSS-induced colitis in mice. METHODS: Splenocytes were isolated from male Balb/c mice. Colitis was induced in rodents, with either trinitrobenzene sulfonic acid or dextran sulfate sodium (DSS). Colonic strips were collected 5 or 6 days after colitis induction. Splenocytes or colonic strips were exposed to CDDO-Im (0.5-2 µM) concomitantly with IL-23 + IL-1ß. Supernatants were collected after 48 or 24 h, and IL-17 was measured by ELISA. Using a DSS colitis model, mice were dosed orally with vehicle or CDDO-Im (20 mg/kg) over a 5-day period. Subsequently, various parameters of colitis were determined on study day 6. RESULTS: Ex vivo treatment with CDDO-Im inhibited IL-17 secretion from splenocytes and colonic strips. The IC50 values were ≤0.62 µM. In vivo, CDDO-Im improved the altered colonic histology, and cytokine (IL-6, and IL-17) contents. Colonic STAT3 activation was also significantly reduced by CDDO-Im treatment. CDDO-Im attenuated IL-17 secretion in ex vivo models of inflammation. In vivo, histological and biochemical parameters of colitis were improved in CDDO-Im treated mice. CONCLUSION: CDDO-Im has a unique pharmacological profile, which supports further testing in animal models of IBD.

Bacillus coagulans GBI-30, 6086 limits the recurrence of Clostridium difficile-Induced colitis following vancomycin withdrawal in mice.

BACKGROUND: Recently, we found that the probiotic strain Bacillus coagulans GBI-30, 6086 (GanedenBC30) improved indices of Clostridium difficile (C. difficile)-induced colitis in mice (Fitzpatrick et al., Gut Pathogens, 2011). Our goal was to determine if BC30 could also prevent the recurrence of C. difficile-induced colitis in mice, following initial treatment with vancomycin. During study days 0 through 5, mice were treated with antibiotics. On day 6, the C. difficile strain VPI 10463 was given by oro-gastric gavage at ≈ 5x104 CFU to induce colitis. Mice were treated on study days 6 to 10 with vancomycin (50 mg/kg) (vanco) or vehicle (saline) by gavage. On days 10 to16, mice were dosed by gavage with saline vehicle or BC30 (2 x 109 CFU per day). Mice were monitored for mortality, weight loss and diarrhea. On study days 14, 16 and 17, stools and colons were collected for analyzing other parameters of colitis. RESULTS: The mean stool consistency score in Vehicle/C.difficile/Vanco mice increased from 0.4 (day 10) to a range of 1.1 to 1.4 (days 14 to 17), indicating the recurrence of colitis. On days 13 through 17, the stool consistency scores for the vancomycin/BC30 mice were significantly lower (p< 0.05) than for the vancomycin/vehicle cohort of animals. On day 17, 88.9% of mice treated with BC30 had normal stools, while this value was 0% with vehicle treatment (p value = 0.0004). Colonic myeloperoxidase (Units/2 cm colon) was significantly (p < 0.05) reduced from 4.3 ± 0.7 (Vehicle/C.difficile/Vanco) to 2.6 ± 0.2 (BC30/C. Difficle/Vanco). The colonic histology score and Keratinocyte derived-chemokine level in the colon were also lower in BC30 treated mice. SUMMARY: In BC30-treated mice, there was evidence of better stool consistency, as well as improved biochemical and histological indices of colitis, following initial treatment of animals with vancomycin. CONCLUSION: BC30 limited the recurrence of CD-induced colitis following vancomycin withdrawal in mice.

Vidofludimus inhibits colonic interleukin-17 and improves hapten-induced colitis in rats by a unique dual mode of action.

Vidofludimus (Vido) is a novel oral immunomodulatory drug that inhibits dihydro-orotate dehydrogenase and lymphocyte proliferation in vitro. Vido inhibits interleukin (IL)-17 secretion in vitro independently of effects on lymphocyte proliferation. Our primary goal was to evaluate the in vivo effects of Vido on IL-17 secretion and the parameters of trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. To further delineate the mechanism of action for Vido, rats were dosed concomitantly with uridine (Uri). Young Wistar rats received a 150-µl enema of either phosphate-buffered saline (PBS) or TNBS on study day 1. The ex vivo effects of Vido on 24-h colonic IL-17 secretion were determined by using colonic strips from PBS- or TNBS-treated rats. Some rats were dosed with vehicle, Vido, or Vido + Uri for 6 days. On day 6, the parameters of colitis were determined from colonic tissue. These parameters included macroscopic, histological, and transcription factor measurements, IL-17 production, and numbers of CD3+ T cells. Ex vivo Vido completely blocked IL-23 + IL-1ß-stimulated secretion of IL-17 by colonic strips. In vivo Vido treatment alone most effectively reduced macroscopic and histological pathology and the numbers of CD3+ T cells. In contrast, similarly reduced nuclear signal transducer and activator of transcription 3 (STAT3) binding and IL-17 levels were observed from animals treated with Vido alone and Vido + Uri. Vido improves TNBS-induced colonic inflammation by a unique dual mode of action: 1) inhibiting expansion of colonic T lymphocytes, and 2) suppressing colonic IL-17 production, which is independent from the control of T-lymphocyte proliferation, by inhibition of STAT3 and nuclear factor-κB activation.

Bacillus Coagulans GBI-30 (BC30) improves indices of Clostridium difficile-Induced colitis in mice.

BACKGROUND: Probiotics have beneficial effects in rodent models of Clostridium difficile (C. diffiicle)-induced colitis. The spore forming probiotic strain Bacillus Coagulans GBI-30, 6086 (BC30) has demonstrated anti-inflammatory and immune-modulating effects in vitro. Our goal was to determine if BC30 improved C. difficile-induced colitis in mice. Starting on study day 0, female C57BL/6 mice were dosed by oro-gastric gavage for 15 days with vehicle (saline) or BC30 (2 × 109 CFU per day). Mice in the C. difficile groups received an antibiotic mixture (study days 5 to 8 in the drinking water), and clindamycin (10 mg/kg, i.p., on study day 10). The C. difficile strain VPI 10463 was given by gavage at 104 CFU to induce colitis on day 11. On day 16, stools and colons were collected for further analyses. RESULTS: All mice treated with BC30 survived on study day 13, while two mice treated with vehicle did not survive. On day 12, a significant difference (p = 0.0002) in the percentage of mice with normal stools (66.7%) was found in the BC30/C. difficile group, as compared to the vehicle/C. diffcile group (13.0%). On study day 16, 23.8% of mice treated with BC30 had normal stools, while this value was 0% with vehicle treatment (p value = 0.0187). On this day, the stool consistency score for the BC30/C. difficile group (1.1 ± 0.2) was significantly lower (p < 0.05) than for the vehicle/C. difficile cohort (1.9 ± 0.2). BC30 modestly attenuated the colonic pathology (crypt damage, edema, leukocyte influx) that was present following C. difficile infection. Colonic MIP-2 chemokine contents (pg/2 cm colon) were: 10.2 ± 0.5 (vehicle/no C. difficile), 24.6 ± 9.5 (vehicle/C. difficile) and 16.3 ± 4.3 (BC30/C. difficle). CONCLUSION: The probiotic BC30 improved some parameters of C. difficile-induced colitis in mice. BC30 prolonged the survival of C. diffiicle infected mice. Particularly, this probiotic improved the stool consistency of mice, in this infectious colitis model.

4SC-101, a novel immunosuppressive drug, inhibits IL-17 and attenuates colitis in two murine models of inflammatory bowel disease.

BACKGROUND: Dihydroorotate dehydrogenase (DHODH) is a key enzyme involved in pyrimidine biosynthesis. DHODH is a known target for the treatment of autoimmune diseases. 4SC-101 is a novel immunosuppressive drug that inhibits DHODH. A goal of our study was to examine the in vitro effects of 4SC-101 on IL-17 production by mononuclear cells. In addition, we evaluated the efficacy of 4SC-101 against acute TNBS (2,4,6-tritrobenzene sulfonic acid) and chronic dextran sodium sulfate (DSS)-induced colitis in mice. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy human donors were used to evaluate cellular proliferation and cytokine (IL-17, TNF-α) production. The oral effects of 4SC-101 (100 or 200 mg/kg) were examined following induction of chronic colitis by the administration of 3% DSS (4 cycles) to Balb/c mice. Morphometric and histological indices of colitis were evaluated as indicators of drug efficacy. 4SC-101 was also administered for 6 days after the intracolonic administration of TNBS (20 mg in 50% ethanol) to female Balb/c mice. The colons were analyzed for overall macroscopic damage, ulceration, total length, distal segment weight, MPO activity, and histological pathology as indicators for the effectiveness of 4SC-101. RESULTS: In vitro, 4SC-101 is a potent inhibitor of human DHODH, inhibits lymphocyte proliferation, and uniquely blocks phytohemagglutinin-stimulated IL-17 production by lymphocytes. In vivo, oral administration of 4SC-101 effectively improved both chronic DSS and acute TNBS colitis in mice. In these colitis models the overall efficacy profile of 4SC-101 was similar to that of dexamethasone. CONCLUSIONS: 4SC-101 is a novel immunosuppressive drug with excellent potential for the treatment of intestinal inflammation.

A new model of chronic hapten-induced colitis in young rats.

AIM AND OBJECTIVE: : Chronic models of inflammatory bowel disease are lacking in preadult rodents. The primary goal of our study was to develop a chronic model of hapten-induced intestinal inflammation and fibrosis in young rats. Second, we aimed to determine the profiles of key Th-1, Th-2, and Th-17 proinflammatory and profibrotic cytokines, during the progression of colitis in young rats. MATERIALS AND METHODS: Chronic hapten-induced colitis was induced by the administration of intracolonic 2,4,6-trinitrobenzene sulfonic acid (TNBS) in young Wistar rats (postnatal days 23, 35, 48, and 59). After 1, 3, or 4 cycles of TNBS, rats were euthanized and the colons were removed for the measurement of macroscopic, histologic, and biochemical parameters of colitis. RESULTS: Young rats developed moderate to severe colitis in the distal colon, without significant morbidity or mortality. Macroscopic severity, histologic pathology, and colonic weights increased progressively with repeated TNBS administration. Cobblestone-like ulceration and fibrosis was evident in the colon, particularly after 4 cycles of TNBS. There was a unique cytokine pattern associated with colitis in young rats. Interleukin (IL)-12 and tumor necrosis factor (TNF)-alpha peaked during the earlier postnatal time points (days 28 and 54) and then declined after repetitive administration of the hapten (day 67). In contrast, IL-13 and IL-17 were consistently elevated after administration of TNBS to the colon of young rats. CONCLUSIONS: A new model of colitis was established in young rats, which has a unique pattern of Th-1, Th-2, and Th-17 cytokine induction. This chronic TNBS model may be useful for studying the development of inflammation and fibrosis in preadult animals.

Effects of the probiotic formulation VSL#3 on colitis in weanling rats.

BACKGROUND: Only a few studies have used models of inflammatory bowel disease (IBD) with weanling animals. Previously, the effects of probiotics have not been assessed in such IBD models. The objectives of our study were 2-fold: to establish a suitable model of dextran sulfate sodium (DSS)-induced colitis in weanling rats and to determine the effects of the probiotic formulation VSL#3 on DSS-induced colitis in weanling animals. MATERIALS AND METHODS: Rats were weaned on postnatal day 21 and administered 2%, 2.5%, or 3% (wt/vol) DSS in drinking water. In subsequent experiments, newly weaned animals were administered vehicle or VSL#3 (0.06, 0.6, or 6 mg) by orogastric gavage. These treatments were given to animals maintained on water (postnatal days 21-28) and then on DSS (postnatal days 28-35). Disease activity indices were determined on a routine basis. On day 35, rats were euthanized. The total colon length was determined. Other parameters of colitis were measured from the distal colon. These parameters included myeloperoxidase (MPO), interleukin (IL)-1beta, inhibitory kappaB-alpha (IkappaB-alpha), and histological assessment of crypt damage and inflammation. RESULTS: DSS 2% was optimal for inducing colitis in weanling rats without significant morbidity. VSL#3 treatments improved various parameters of 2% DSS-induced colitis in weanling rats. The 0.6- and 6-mg doses of VSL#3 were most effective for attenuating this colitis. CONCLUSIONS: The probiotic formulation VSL#3 improved DSS-induced colitis in weanling rats. This improvement of colitis involved changes in colonic IkappaB-alpha, IL-1beta, and MPO, which are suggestive of immune modulation by VSL#3.

Enhanced intestinal expression of the proteasome subunit low molecular mass polypeptide 2 in patients with inflammatory bowel disease.

PURPOSE: Low molecular mass polypeptide 2 is an inducible immunoproteasome subunit. The expression of low molecular mass polypeptide 2 has not been examined in the intestine of patients with inflammatory bowel disease. This study was designed to determine whether the intestinal expression of low molecular mass polypeptide 2 was enhanced in a group of patients with inflammatory bowel disease compared with a group of control patients without inflammatory bowel disease. Moreover, we examined the association between low molecular mass polypeptide 2 expression and histologic pathology in these patients. METHODS: Twenty-one patients participated in the study. These included six control subjects without inflammatory bowel disease, eight patients with ulcerative colitis, and seven patients with Crohn's disease. Intestinal low molecular mass polypeptide 2 expression was evaluated by immunohistochemistry, as well as by Western blot. Histology scores (0-40 severity scale) were determined on the same sections of intestine as those used for low molecular mass polypeptide 2 histochemistry. RESULTS: By immunohistochemistry, low molecular mass polypeptide 2 expression was significantly enhanced (P < 0.05 vs. control subjects) throughout visibly diseased areas of colon, rectum, and ileum from patients with inflammatory bowel disease. Low molecular mass polypeptide 2 expression also was increased in macroscopically normal intestine from patients with inflammatory bowel disease compared with normal tissue from control subjects. There was a significant correlation (P < 0.0001) between low molecular mass polypeptide 2 expression and histologic pathology in our patients. Western blot results confirmed that low molecular mass polypeptide 2 expression was enhanced in patients with ulcerative colitis (3.1-fold) and in patients with Crohn's disease (3.5-fold). CONCLUSIONS: Intestinal low molecular mass polypeptide 2 expression is significantly increased in inflammatory bowel disease. The association between intestinal low molecular mass polypeptide 2 expression and histologic pathology suggests that this proteasome subunit plays a role in the pathogenesis of inflammatory bowel disease.

Dextran sulfate sodium-induced colitis is associated with enhanced low molecular mass polypeptide 2 (LMP2) expression and is attenuated in LMP2 knockout mice.

Low molecular mass polypeptide 2 (LMP2) is an inducible proteasome subunit. Our goals were to examine LMP2 expression in mice with dextran sulfate sodium (DSS)-induced colitis and to evaluate colitis in LMP2 knockout (LMP2-/-) mice. Mice were given 2.5% DSS in the drinking water. On day 0, 2, 4, or 6 after DSS treatment, LMP2 expression was determined in the distal colon by western blot and immunohistochemistry. Parameters of colitis were measured in LMP2-/- mice or wild-type mice. LMP2 expression was enhanced in the colon of DSS-treated mice at all time points. Symptoms of DSS-induced colitis were always lower in LMP2-/- mice. Normalized histology scores and colonic IL-1ss levels increased over the 6-day study period in wild-type mice. These parameters were significantly reduced in LMP2-/- mice that consumed DSS for 6 days. Enhanced LMP2 expression contributes to the pathogenesis of DSS-induced colitis in mice.